PROTEIN ROTATIONAL DIFFUSION AND LIPID PROTEIN INTERACTIONS IN RECOMBINANTS OF BOVINE RHODOPSIN WITH SATURATED DIACYLPHOSPHATIDYLCHOLINES OF DIFFERENT CHAIN LENGTHS STUDIED BY CONVENTIONAL AND SATURATION-TRANSFER ELECTRON-SPIN-RESONANCE

Bovine rhodopsin has been reconstituted in seven different saturated diacylphosphatidylcholine species of odd and even chain lengths from C-12 to C-18 at a lipid/protein ratio (60:1 mol/mol) comparable to that in the native rod outer segment disk membrane. All recombinants were found to be photochemically active, in that optical bleaching produced a temperature- and lipid chain-length-dependent mixture of species absorbing at 480 and 380 nm. Both the rotational diffusion of rhodopsin and lipid-protein interactions in the various recombinants were studied by saturation transfer and conventional electron spin resonance spectroscopy of spin-labeled rhodopsin and of spin-labeled phosphatidylcholine, respectively. In the fluid lipid phase, the rotational diffusion rate of rhodopsin was found to be dependent on the lipid chain length of the different recombinants in a nonmonotonic manner. The diffusion rate in dilauroylphosphatidylcholine was found to be very slow, indicating extensive protein aggregation, whereas that in dipentadecanoylphosphatidylcholine was rapid (effective correlation time ca. 7-mu-s), consistent with the presence of monomeric protein. For recombinants with longer lipid chain lengths, the rotational diffusion rate again decreased, indicating the presence of di- or oligomeric protein. The fraction of lipid motionally restricted at temperatures in the fluid phase was also dependent on the chain length of the phosphatidylcholine used in the reconstitution. For recombinants with dimyristoyl- and dipentadecanoylphosphatidylcholine, the number of lipids estimated to be associated with the hydrophobic surface of the protein (ca. 22 lipids/protein) was consistent with the presence of monomeric protein of intramembranous diameter of ca. 30 angstrom. In recombinants with dilauroylphosphatidylcholine, the fraction of motionally restricted lipid was greater, indicating that up to 22 lipids/protein were trapped in the protein aggregates, in addition to those motionally restricted at the hydrophobic perimeter of the protein. For recombinants with phosphatidylcholines of chain lengths longer than C-15, the fraction of motionally restricted lipid was reduced, to a level beyond that of detectability in the case of distearoylphosphatidylcholine. This reduction in hydrophobic surface available to lipids is again consistent with aggregation of the protein in recombinants of longer lipid chain length, indicating extensive protein aggregation for the C-18 chain length. The effects of phospholipid chain length on lipid selectivity at the protein interface are also considered.